Different carbon sources were investigated for overproduction penicillin G acylase from Alcaligenesfaecalis in recombinant Escherichia coli strains. The results indicated that the enzyme was optimally produced with 45 g/l of dextrin, and about 43,385 and 79,880 U/l for the highest enzyme activities were obtained in batch cultivations of shaken flasks and a 3.71 bioreactor, respectively. Active site titration and SDS-PAGE electrophoretic analysis demonstrated that the maximum yield of the active enzyme was 2.54 g/l, which was about 40% of total soluble proteins. The highest specific activity of A. jaecalis penicillin G acylase obtained was above 10 U/mg protein, and there was almost no plasmid lost in the whole batch cultivations. Furthermore, the cultivation process was relatively simple and suitable for large-scale production. (c) 2007 Elsevier Inc. All rights reserved.